Visualization and Quantitative Analysis of Genotoxin-Induced PARP1/PARP2 Activation in Cells Using a Fluorescent Fusion Protein-Based Reporter

Summary

Here, we describe a method for cell-based visualization, subcellular localization, and quantitative analysis of poly(ADP-ribose) (PAR) enriched foci in fixed mammalian cells. This assay enables the quantification of sites of DNA damage-induced PARP activation, including that associated with base excision repair or single-strand break repair, in the nuclei of genotoxin-exposed cells.

Abstract

Poly(ADP-ribose) (PAR) is a polymer of ADP-ribose synthesized by four members of the ADP-ribose polymerase family of enzymes—PARP1, PARP2, PARP5a, and PARP5b. However, only PARP1 and PARP2 synthesize PAR in response to DNA breaks. PAR is defined as a protein post-translational modification, but it is also shown to exist as a DNA or RNA modification. Levels of PAR are further regulated by PARG, a PAR glycohydrolase that, together with PARP1 and PARP2, modulates the cellular level of DNA damage-induced PAR. The dynamic synthesis and degradation of PAR is critical to its regulatory role in DNA repair, and the DNA damage response, which in turn affects chromatin reorganization, replication, transcription, and cell death. PARP1/PARP2 activation and the accumulation of PAR can be considered sites of ongoing base excision repair or DNA single-strand break repair; however, numerous PARP1/PARP2 activators are also associated with replication stress and other DNA metabolic processes. Once formed, PAR chains facilitate the recruitment of DNA repair and DNA damage response (DDR) factors to sites of DNA damage or genomic insult via their PAR-binding domains (PBDs). Ten different PBDs recognize various regions of the PAR molecule, including the PAR binding motif, PAR binding Zinc finger, the WWE domain, and the macrodomain, among other PBDs. To facilitate cellular analysis of PAR dynamics, we used PBDs fused to enhanced green fluorescent protein (EGFP) to optimize cell-based quantitation of PAR foci. We describe an assay that uses a fragment of RNF146 encoding the PBD/WWE domain, linked to EGFP, to visualize and quantify PAR accumulation at sites of genomic insult and ongoing BER or SSBR. We describe experimental steps, including the production of lentiviral particles, transduction of the target cell line, treatment of mammalian cells to induce genomic DNA damage, acquisition of confocal fluorescence micrographs, and semi-automated quantification of the data.

Introduction

This protocol describes the LivePAR assay, which allows visualization and quantitative analysis of poly(ADP-ribose) (PAR) at sites of genomic insult, primarily associated with base excision repair (BER) or single-strand break repair (SSBR), in mammalian cells^1,2,3. BER is a crucial DNA repair pathway that removes damaged or modified bases from DNA (for reviews on BER and PARylation, see^4,5,6,7). BER begins with DNA glycosylases that recognize and excise modified bases⁴, leaving an apurinic/apyrimidinic (AP) site, for example, methyl purine glycosylase (MPG), which excises methylated bases^8,9, or 8-oxoguanine glycosylase (OGG1), which excises 8-oxoG^9,10. AP sites are then cleaved by AP endonuclease 1 (APE1)^11,12,13, creating a nick in the DNA backbone. A polymerase, namely DNA polymerase β (Polβ)^14,15, tailors and fills the gap using the undamaged strand as a template, and finally, DNA ligases seal the repaired strand^16,17, for example, DNA ligase I or DNA ligase III.

Key players in BER and SSBR are poly(ADP-ribose) polymerases 1 and 2 (PARP1/PARP2)^18,19,20, enzymes that rapidly detect DNA strand breaks during BER or SSBR. Upon detecting these breaks, PARP1/PARP2 catalyzes the addition of ADP-ribose polymers to itself and other proteins—a process called PARylation (for review, see⁶)—by hydrolyzing NAD⁺. This creates a dynamic protein scaffold (PAR) that recruits other DNA repair factors to the site of damage, enhancing the fidelity of DNA repair. To manage this dynamic PAR scaffold, several proteins contribute to the hydrolysis and removal of PAR chains, including ADP-ribose-acceptor hydrolase 3 (ARH3), terminal ADP-ribose glycosyl hydrolase 1 (TARG1), mono-ADP-ribosyl hydrolase 1/2 (MacroD1/2), and poly(ADP-ribose) glycohydrolase (PARG)^{6,21,22,23,24,25,26}. However, PARG is primarily responsible for this process^21,27 and plays a crucial role in the DNA damage response (DDR)^28,29.

PARG is therefore critically linked to the function of PARP1 and PARP2. While PARP1 and PARP2 create the PAR polymers, PARG acts as a "clean-up" enzyme, hydrolyzing these polymers and effectively reversing PARylation^30,31. This process is essential for completing both BER and SSBR and for allowing PARP1/PARP2 to be recycled to respond to new DNA damage. Consequently, PARP1, PARP2, PARylation, and PARG work in a coordinated manner, ensuring efficient and reversible DNA repair through BER and SSBR^4,6.

The extent of PARylation can be influenced by various factors and plays an essential role in the overall DNA damage response³². These factors include exposure to genotoxins that add adducts to DNA bases, such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), hydrogen peroxide (H₂O₂), or tert-butyl hydroperoxide (tBuOOH). Further, the extent of PARylation can be influenced by molecules that modulate the PARylation process, such as dihydronicotinamide riboside (NRH), a precursor to NAD⁺, which enhances PARP-activation by increasing NAD⁺ levels, or FK866, an inhibitor of NAMPT, which can decrease NAD⁺ levels and negatively regulate PARP-activation³. Finally, genetic mutations or small-molecule inhibition of enzymes such as PARP1/PARP2, PARG, or other DNA repair proteins (e.g., defects in BER or homologous recombination factors such as BRCA1/BRCA2) can alter PARP activation^33,34,35,36.

The LivePAR assay makes use of a fragment of the ring finger protein 146 (RNF146) that encodes a WWE domain (amino acids 100–182), linked to an enhanced green fluorescence protein (EGFP)³. The WWE domain is a globular domain named after the conserved residues tryptophan (W) and glutamate (E) in its binding site³⁷. WWE domains bind to iso-ADP-ribose, the smallest internal structural unit within PAR chains^38,39. RNF146 is an E3 ubiquitin ligase that recognizes iso-ADP-ribose^38,40 and is reported to target proteins involved in BER (XRCC1, DNA ligase III, and PARP1) for proteasomal degradation⁴¹. Linking this WWE domain to EGFP enables visualization and detection of genotoxin-induced PARylation sites in mammalian cells. In this protocol, we describe the generation of stable cell lines expressing the LivePAR reporter (Figure 1A), the induction of DNA damage to activate PARP1/PARP2, the production of PAR, and the acquisition and analysis of confocal fluorescence microscopy data (Figure 1).

The pMD2.g(VSVG) plasmid provides the viral envelope glycoprotein (VSVG), which expands the range of cells the virus can infect compared to the native lentivirus envelope protein. The pRSV-REV plasmid provides the regulatory element (REV) protein, which is a crucial transactivator that binds to the viral RNA and activates the transcription of the viral genes; it is essential for efficient viral replication. The pMDLg/pRRE plasmid provides several key internal packaging components, namely gag, which encodes the matrix (MA), capsid (CA), and nucleocapsid (NC) proteins, pol, which encodes the enzymes needed for viral replication and integration into the host cell genome, and the RNA regulatory element (RRE), which is an RNA element that enhances the efficiency of viral RNA translation and genomic RNA packaging. The pLV-EF1A-LivePAR-Hygro plasmid (Figure 1A) contains cDNA coding for the enhanced green fluorescence protein (EGFP) that is fused to the C-terminus of the WWE domain of RNF146 (amino acids 100–182), a protein domain that binds to poly(ADP-ribose) chains, and the vector also encodes a hygromycin resistance cassette. The virus is isolated from the cell culture supernatant and can be stored at -80 ˚C or used immediately for transduction (Figure 1B).

Protocol

1. Lentivirus production

NOTE: This protocol employs a third-generation lentiviral packaging system^42,43 to generate cells expressing a genetically encoded PAR reporter. Lentiviral particles are produced in 293FT cells using a four-plasmid system comprising envelope, regulatory, and packaging components, together with the pLV-EF1A-LivePAR-Hygro construct encoding an EGFP-tagged WWE domain of RNF146, which binds poly(ADP-ribose) chains. The resulting virus is used to generate stable cell lines for visualization and quantification of PAR accumulation.

1. Culture low passage 293FT cells in DMEM containing 10% FBS and 1% Pen/Strep in a humidified atmosphere at 37 ˚C and 5% CO₂. Ensure that the 293FT cells have not been in culture for more than 3 weeks.
   NOTE: We routinely use 293FT cells ranging in passage numbers between 3 and 10. 293FT cells were transformed and immortalized by the SV40 large T antigen. These cells have a high transfection efficiency, which is essential for recombinant protein production and gene expression studies. They respond well to both lipid-based transfection and viral transduction methods.
2. Plate 1.7 × 10⁵ 293FT cells onto a 60 mm tissue culture dish in a final volume of 5.5 mL of culture media on day 1. Return cells to the incubator for 24 h.
3. On day 2 (the day of transfection), add 375 µL of FBS- and Pen/Strep-free DMEM medium to a 1.5 mL sterile microcentrifugation tube. Then, add 10 µL of a lipid-based transfection reagent, 4 µg of the pLV-EF1A-LivePAR-Hygro plasmid, 2 µg of the pMD2.g(VSVG) plasmid, 2 µg of the pRSV-REV plasmid, and 2 µg of the pMDLg/pRRE plasmid. Tap the microcentrifugation tube gently to mix the media, transfection reagent, and plasmid DNA (avoid vortexing). Incubate for 15–30 min at room temperature to allow for DNA/transfection reagent complex formation.
4. Add the plasmid/transfection reagent mix to the 60 mm dish containing the 293FT cells to be transfected without removing the media, dropwise. Return the cells to the incubator for 48 h.
   NOTE: This period allows for the production and release of the lentiviral particles into the growth media.
5. On day 4, collect the culture media from the transfected 293FT cells to isolate the lentivirus particles. Filter through a sterile 0.45 µm filter to separate cell debris from the lentiviral particles.
6. Determine the lentivirus titer with available reagents (optional).
   NOTE: This method generally yields 10⁵ to 10⁶ infectious units per mL (IFU/mL), which correlates to an approximate multiplicity of infection (MOI) of ~5 when used as outlined in the transduction step below.
7. Aliquot the media containing the lentivirus particles into four sterile cryovials (1.0 mL per tube) and store at -80 ˚C.

2. Transduction

NOTE: This section outlines the lentiviral transduction protocol used to introduce a transgene into cell lines (Figure 1B). It has been consistently effective in our lab^{1,3,33,37,44,45}.

1. Culture a cell line of choice (low passage number) in the appropriate media in a humidified atmosphere at 37 ˚C and 5% CO₂.
   NOTE: Start with an early passage of the cell type needed. A new vial of cells must be thawed and passaged at least once before seeding for transduction.
2. On day 1, seed 1 × 10⁵–1.5 × 10⁵ cells per well, onto each well of a 6-well plate in 2 mL of growth media. Return the cells to the incubator for 24 h.
3. On day 2 (the day of transduction), ensure that the cells are between 20% and 40% confluent. Add 1 mL of virus, 1 mL of growth media, and 2 µL of polybrene (2 mg/mL stock) to a 15 mL conical tube and mix gently by inversion (avoid vortexing).
   NOTE: Polybrene is used during lentivirus transduction to prevent electrostatic repulsion between the virus particles and the cell membrane. As polybrene is positively charged, it interacts with the negatively charged viral glycoproteins, neutralizing their charge. Furthermore, polybrene binds to glycosaminoglycans in the cell culture medium, preventing their interaction with the virus. Polybrene, therefore, greatly enhances transduction efficiency.
4. Remove the growth media from the 6-well plate and add the virus/media/polybrene mix to each well. Place the cells with the transduction mix in an incubator containing a humidified atmosphere at 32 ^°C and 5% CO₂ for 16–18 h.
   NOTE: Incubating cells at 32 °C overnight during lentiviral transduction is a common, though not essential, practice that significantly increases viral infection efficacy. Lowering the temperature slows cellular metabolism, thereby reducing cellular defenses and increasing susceptibility to viral infection.
5. On day 3, replace the media of the virus-transduced cells with fresh media and move them to a 37 °C incubator. Incubate for 48 h.
   NOTE: These 2 days are sufficient for the cells to produce the RNF146(100–182)-EGFP transgene and become resistant to hygromycin.
6. To develop the resulting polyclonal stable cell lines expressing the RNF146(100–182)-EGFP transgene, next start hygromycin selection after 2 days of incubation as indicated in step 2.5.
   1. Alternatively, expand this population of transduced cells in the absence of hygromycin and use for PAR analysis over the course of the next 10–14 days (transient transduction), as described previously¹.
7. Transfer the cells onto a 100 mm plate once they grow in the presence of hygromycin and expand as much as required.
8. Prepare cryo stocks of the transduced cells for future use.
9. Validate the expression of the RNF146(100–182)-EGFP transgene by confocal microscopy as outlined in section 3. The RNF146(100–182)-EGFP transgene will be expressed throughout the cell.
10. Validate the expression of the RNF146(100–182)-EGFP transgene by immunoblotting, as described elsewhere^1,37.

3. Confocal microscopy and analysis

NOTE: This section of the protocol details the preparation of coverslips, the seeding of the LivePAR-expressing cells, exposure to treatments (tBuOOH, MNNG, PARGi, PARPi, and/or NRH), and preparation of fixed cells on microscope slides for confocal imaging and PAR foci analysis (Figure 1C). It also describes the use of ImageJ software and a custom macro for the semi-automated quantification of PAR foci. This protocol can be used to investigate the influence of various factors (e.g., small molecules, BER protein knockouts) on DNA damage-dependent PARylation and has been successfully employed in our lab^34,37.

1. Coverslip preparation
   1. Soak 20 × 20 mm coverslips for 20 min in diethyl ether in a glass container in a fume hood.
      NOTE: Use a fume hood when working with diethyl ether due to its flammability and potential hazards.
   2. Remove the diethyl ether and wash the coverslips in decreasing concentrations of ethanol [100%, 70%, 50%, then 0% (ddH₂O)].
   3. Remove the water and soak the coverslips for 20 min in 1 N HCl with gentle agitation.
   4. Remove the HCl and wash the coverslips 3x with ddH₂0.
   5. Store the coverslips in 70% ethanol at 4 °C until use.
2. Plating of cells
   1. Place two 20 × 20 mm sterile coverslips in a 60 mm cell culture dish next to each other. Prepare as many dishes as required for the experiment. Ensure that all the ethanol has evaporated before adding any cells to the dish containing the coverslips. Wash away any residual ethanol by adding sterile PBS to the dish for 10–15 min; remove PBS before seeding the cells.
   2. Seed 200,000 LivePAR-expressing cells per cell culture dish. Allow the cells 24–36 h to adhere to the coverslips, condition the medium, and start replicating (growing).
3. Induction of PAR foci
   1. Expose the LivePAR-expressing cells to 100 µM NRH, 10 µM PARGi, and 10 µM MNNG or 40 µM tBuOOH. In addition, to test for PARP1/PARP2-dependent PAR formation, inhibit PARP1/PARP2 by adding 10 µM PARPi. Add compounds directly to the media containing the cells on the coverslips and gently swirl the media in the cell culture dishes to distribute the compounds. Incubate the 60 mm dishes at 37 ˚C for 60–90 min.
      NOTE: NRH is used to ensure that sufficient levels of cellular NAD⁺ is available for optimal PARP1/PARP2-mediated PARylation^33,46,47. PARGi is used to prevent the PAR chains synthesized by PARP1/PARP2 from being broken down by PARG. MNNG or tBuOOH is used to induce the DNA base modifications to initiate BER, which leads to the activation of PARP1/PARP2. PARPi is used to confirm that the signal (foci) results from PARP1/PARP2 activation.
4. Fixation of cells and mounting of the coverslips
   1. Remove the medium and wash with PBS.
      NOTE: Washing once or twice with PBS is important, as the medium used to culture cells contains a high concentration of serum proteins. Washing with PBS also removes any residual MNNG or tBuOOH still in the media.
   2. Prefix in 4% formaldehyde in PBS for 15 min at room temperature. Wash cells 3x with PBS.
   3. Fix by adding 3 mL of cold (-20 ^°C) methanol: acetone (7:3) to the cells. Place dishes at -20 ^°C for 9 min.
   4. Remove methanol: acetone. Wash 3x with PBS.
   5. Pipette 15 µL of anti-fade medium containing DAPI on the glass slides. Gently mount the coverslips onto the spot of mounting medium with the cells facing inward, minimizing bubbles.
   6. Center, secure, and seal the sides of the coverslip to the glass slide by applying a small amount of clear topcoat nail enamel to the sides of the coverslip. Place the slides in the dark to let the topcoat nail enamel dry (~10 min). Store slides in the dark at 4 ˚C until ready to image.
5. Imaging cells
   1. Image all slides at least 10x at 63× magnification, using a confocal microscope. Detect PAR foci using the 488 nm laser and nuclear staining (DAPI) using the 405 nm laser (for example, see Figure 2).
6. Analyzing confocal images
   1. Create a text file containing the following text and name it “LivePAR_Macro”. This macro facilitates the “automatic” quantification of PAR foci in the nuclei of the LivePAR-expressing cells:
      ​macro "DAPI positive staining [1]" {
      tit=getTitle();
      run("Duplicate...", "title=DAPI duplicate");
      selectWindow("DAPI")
      run("8-bit");
      run("Split Channels")
      selectWindow("C2-"+"DAPI");
      close();
      selectWindow("C1-"+"DAPI");
      run("Gaussian Blur...", "Sigma=3");
      setAutoThreshold("Default");
      run("Make Binary");
      run("Invert")
      run("Fill Holes")
      run("Watershed")
      run("Analyze Particles...", "size=50.00-Infinity circularity=0.00-1.00 show=Outlines add exclude include in_situ");
      rename("DAPI_"+tit);
      selectWindow("DAPI_"+tit);
      close();
      }
      macro "PAR [2]" {
      setBatchMode(true);
      run("8-bit");
      t=getTitle();
      run("Duplicate...", "title=GB1 duplicate");
      run("Duplicate...", "title=GB20 duplicate");
      selectWindow("GB20")
      run("Gaussian Blur...", "radius=20 stack");
      selectWindow("GB1");
      run("Gaussian Blur...", "radius=1 stack");
      imageCalculator("Subtract create stack", "GB1","GB20");
      selectWindow("GB1");
      close();
      selectWindow("GB20");
      close();
      selectWindow("Result of GB1");
      rename("Blank_GB1-GB20_"+t);
      title = getTitle();
      run("Split Channels")
      selectWindow("C1-"+title);
      close();
      selectWindow("C2-"+title);
      setThreshold(90,255) //the 90 value can be changed to account for experimental variability
      run("Analyze Particles...", "size=0.00-200 circularity=0.00-1.00 show=Masks summarize");
      close ("C2-"+title);
      selectWindow("Mask of C2-"+title);
      close ();
      setBatchMode(false);
      }
   2. Download and install ImageJ onto the computer (https://imagej.net/ij/download.html).
   3. Open ImageJ and install the macro (text file: LivePAR_Macro) by clicking on Plugins | Macros | Install.
   4. Open all confocal files in ImageJ and save them as *.tif files to preserve the original files.
   5. Open a spreadsheet document and save it as [date]_[Cell Line]_Foci Analysis.
   6. Analyze one *.tif file at a time. Press 1 to find the nuclei; when a window titled ROI Manager opens, select all entries in the window and press add to overlay the nuclei area over the original image. Delete incorrectly identified nuclei. Draw the nucleus using the freehand selection tool and press 2 to quantify PAR foci.
      NOTE: Do not include nuclei that are not fully within the frame.
   7. Select each nucleus in the ROI manager one by one and press 2. A count will appear indicating the number of foci within the nucleus selected. Once the foci in all the nuclei of the file have been scored, copy and paste the information into the open spreadsheet document.
   8. Repeat steps 3.6.6 and 3.6.7 with all *.tif files.
   9. Plot PAR foci per nucleus in the software of choice (e.g., see Figure 3).

Results

Cells expressing the LivePAR probe can be generated by producing a lentivirus encoding the RNF146(100-182)-EGFP fusion protein (Figure 1A). Cells can be evaluated after transient expression for up to two weeks after transduction or as stable cell lines following selection in hygromycin (Figure 1B)¹. Following genotoxin treatment, DNA damage-induced PAR foci can be visualized within the nucleus and, once fixed, imaged by confocal fluoresce...

Discussion

The LivePAR assay was developed to detect sites of nuclear genomic DNA damage where PARP1/PARP2 is actively synthesizing PAR chains. To accomplish this, a fragment of RNF146 encoding the WWE domain (amino acids 100–182) was fused to EGFP^1,2,3,37. Additional PAR probes we have described encode EGFP fused to amino acid residues 1 to 166 of DTX4, amino acid residues 22 to 112 of PARP11, and ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents, Chemicals, and Peptides
0.45 µm Durapore steriflip filters	Sigma-Aldrich	Cat# SE1M003M00
20 x 20 mm Microscope coverslips	Thermo Fisher Scientific	Cat# 22-170-371
Acetone	Thermo Fisher Scientific	Cat# A18-1
Cell culture dishes, 100 mm	VWR	Cat# 25382-166
Cell culture dishes, 60 mm	Fisher Scientific	Cat# 430166
Cell culture plates, 6-well	VWR	Cat# 62406-161
Cryovials, 2 mL	VWR	Cat# 89499-722
Diethyl ether	Thermo Fisher Scientific	Cat# 60-29-7
Dimethyl sulfoxide	Thermo Fisher Scientific	Cat# BP231-1
Disposable sterile filters, 0.45 µm	Sigma-Aldrich	Cat# SE1M003M00
DMEM	Corning	Cat# 15-017-CV
Ethanol, 200 proof	Decon Labs	Cat# 2701
Glass microscope slides	Thermo Fisher Scientific	Cat# 12-544-4
Heat-inactivated fetal bovine serum	Atlanta Biologics	Cat# S11150
Hydrochloric acid	Thermo Fisher Scientific	Cat# A144-212
Hygromycin	Thermo Fisher Scientific	Cat# 10687010
Large Kim Wipes	Kimberly-Clark	Cat# 34256
Lenti-X GoStix Plus Kit	Takara	Cat# 631280
Methanol	Thermo Fisher Scientific	Cat# A452SK-4
MNNG	Molport	Cat# N493990
NRH (1-[(2R,3R,4S,5R)-3,4-Dihydroxy-5-(hydroxymethyl) tetrahydrofuran-2-yl]-4H-pyridine-3-carboxamide)	Marie Migaud or  NuChem Sciences Inc
PARG inhibitor (PDD00017273)	Sigma-Aldrich	Cat# SML1781
PARPi inhibitor (Veliparib, ABT-888)	MedChemExpress	Cat# HY-10129
Penicillin/streptomycin	Gibco	Cat# 15140-122
Polybrene	Sigma-Aldrich	Cat# TR-1003-G
tBuOOH	Thermo Fisher Scientific	Cat# 180345000
TransIT-x2 (Transfection reagent)	Mirus	Cat# MIR-6006
Triton-X100	VWR	Cat# 0694
Trypsin-EDTA	Thermo Fisher Scientific	Cat# 25200-056
Vectashield (H-1000) anti-fade mounting medium	Thermo Fisher Scientific	Cat# NC1695563
Cell lines
293-FT	Thermo Fisher Scientific	Cat# R70007
(derived from human embryonal kidney cells transformed with the SV40 large T antigen)	Thermo Fisher Scientific	Cat# R70007
LN428	Generous gift from	N/A
(Human glioblastoma tumor cell line)	Dr. Ian Pollack (University of Pittsburgh)	N/A
RPE-1	ATCC	Cat# CRL-4000
(human hTERT-immortalized retinal pigment epithelial cells)	ATCC	Cat# CRL-4000
U2OS	ATCC	Cat# HTB-96
(Human osteosarcoma cell line)	ATCC	Cat# HTB-96
Recombinant DNA
pMDLg/pRRE	Lab stock# 253	Addgene, Cat# 12251
pRSV-Rev	Lab stock# 254	Addgene, Cat# 12253
pMD2.G	Lab stock# 252	Addgene, Cat# 12259
pLV-EF1A-LivePAR-Hygro (LivePAR is the WWE domain of RNF146, amino acid residues 100-182, fused to EGFP; contains a Hygromycin resistance cassette)	Lab stock# 1727	Addgene, Cat# 176063
Software and Algorithms
Adobe Illustrator (for preparation of figures)	Adobe Systems	Version 2024
GraphPad Prism	GraphPad	Version 10.5.0 (Mac OS X)
ImageJ	NIH	https://imagej.net/ij/download.html